Numerous physiological problems, such as a loss in muscle mass, a failure of the immune system, decreases in the maximal synthesis and release of hormones (e.g. insulin or growth hormone), loss of renal function, and decreases in cognitive skills occur with aging. These problems lead to an overall decline in functional capacity. Several models have been advanced to explain these age-related physiological problems. Such models include, for example, increased programmed cell death, i.e. apoptosis; accumulation of oxidant damage; failure of the cell to maintain the telomeres at the ends of the chromosomes; and defects in responding to stress. The observed age-related defects in responding to stress may involve chaperone proteins.
At the cellular level the most important stress proteins are the chaperones. Chaperones play an important role in cellular function. They help to realign proteins into their native state, thereby renaturing damaged proteins and aid the final steps of protein folding by directing newly synthesized proteins into their final, optimal structure. Chaperones also help stabilize the final protein product, such as by the formation of intra- and intermolecular disulfide bonds. One such family of chaperones is known as thiol:protein disulfide oxidoreductases (TPDOs). Studies of the stress proteins and chaperones support the concept that many of the age-related functional declines are associated with decreases in the activity of the chaperone systems. Decreased levels and activity of chaperones can result in increased formation of improperly folded and insoluble masses of proteins.
Insoluble masses, or plaques, of the β-amyloid protein, a 38 to 43 amino acid peptide derived from the amyloid precursor protein, form in the brain of older persons suffering from Alzheimer's disease. Amyloid precursor protein is an intrinsic membrane protein that is synthesized in the endoplasmic reticulum. During synthesis and insertion into the plasma membrane, β-amyloid is cleaved off the amyloid precursor protein and secreted into the intercellular space.
In physiological solutions β-amyloid readily aggregates to form plaques characteristic of Alzheimer's disease. However, Alzheimer's disease is complex and involves more than mere overexpression of the β-amyloid peptide. The neuropathology of Alzheimer's disease is characterized by extensive neuronal cell loss and deposition of numerous senile plaques and neurofibrillary tangles in the cerebral cortex. Although small numbers of classic senile plaques develop in the normal brain with age, large numbers of the plaques are found almost exclusively in Alzheimer's patients.
One study showed that when cerebrospinal fluid is added to β-amyloid, β-amyloid does not aggregate, suggesting that cerebrospinal fluid includes a component that inhibits β-amyloid aggregation. This indicates that cerebrospinal fluid of subjects that are free of Alzheimer's disease may include a component that prevents formation of senile plaques. This component could be a chaperone. Thus, it is desirable to better characterize the role of chaperones in processing of amyloid precursor protein, forming β-amyloid plaques, and Alzheimer's disease. Proper folding or processing of the amyloid precursor protein or β-amyloid may be involved in the etiology of Alzheimer's disease.
Alternatively, a patient with Alzheimer's disease may have a protein that enhances nucleation of β-amyloid plaques. One theory suggests that apolipoprotein E may play a role in Alzheimer's disease. Apolipoprotein E exists in at least 3 allelic forms known as apoE2, apoE3, and apoE4. Evidence indicates that a person who has at least one allele of apolipoprotein E4 (apoE4) is more susceptible to Alzheimer's disease, suggesting that the protein product of apoE4 may play a role in Alzheimer's disease. Moir et al., Biochemistry, 38: 4595-4603 (1999). For example, apoE4 may contribute to the nucleation or formation of β-amyloid plaques by contributing to the aggregation of β-amyloid.
Previously, Alzheimer's disease studies have focused on overproduction of β-amyloid. For instance, many laboratories have investigated the role of proteases involved in cleaving the precursor protein to produce β-amyloid. Yet a number of studies have shown that, with the exception of some rare genetic forms of early onset Alzheimer's disease and the early Alzheimer's disease seen with Down's syndrome, patients with Alzheimer's disease actually have lower concentrations of β-amyloid in their cerebrospinal fluid than age-matched controls. Further, a recent study of transgenic mice having an amyloid precursor protein gene lacking the Kunitz-protease inhibitor domain showed that the increased concentration of β-amyloid cannot be explained by a rise in expression of amyloid precursor protein, which appeared to remain unchanged with age. These studies indicate that β-amyloid levels alone are not enough to explain Alzheimer's disease. Thus, it is desirable to better characterize the role of chaperones and processing of amyloid precursor protein in forming β-amyloid plaques and in Alzheimer's disease. Proper folding or processing of the amyloid precursor protein or β-amyloid may be involved in the etiology of Alzheimer's disease.
Furthermore, at present the only method to detect a propensity for formation of β-amyloid plaque or Alzheimer's disease or the presence of such plaques or disease includes dissection of the brain or culturing of brain cells of the subject. Such invasive procedures are, of course, undesirable for most subjects. This is particularly so, since as outlined above, even after such dissection, it previously would have been unclear how to test for certain factors leading to plaque formation or disease. This demonstrates a need for a method to detect the propensity for or presence of plaques or disease in a living, intact subject.